Aromatic composition consisting of HLA molecules

ABSTRACT

The invention relates to novel aromatic compositions consisting of HLA molecules, which two different potential perfume carriers are assigned. The invention aims at developing novel aromatic compositions consisting of HLA molecules based on the selection of alleles of genes which are relevant from an olfactory viewpoint and subsequent processing thereof. According to the invention, the aromatic compositions are produced by selecting one allele amongst known class I HLA alleles, which differs in at least one characteristic from other alleles of HLA class I molecules and which is present in less than 5% of individuals of the world population; the protein that is coded for by the selected allele undergoes assembly in the presence of β 2 -microglobulin (β 2 m); the formed HLA class I molecules are purified with the bonded peptides and fragmented with the protease, and the odor-active substances resulting from fragmentation are added as individual components or as a mixture to a cosmetic preparation.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new perfume compositions, each assignedto two different potential scent carriers (partners).

2. Description of the Related Art

A plurality of scents of different provenience are known from the stateof the art; these scents have been and will be used to have a positiveinfluence on the presumably inadequate natural body odor by means ofmore or less pleasant smelling essential oils, perfumes or soaps. Inrecent years, it has become known that genetic factors may beresponsible for an individual's natural body odor and thus also for hisor her attractiveness to other partners. Specific studies on volunteers(Am. J. Hum. Genet. 61, 505–51 (1997)) have shown that the choice ofpartners might be influenced by odors by way of polymorphic genes of themajor histocompatibility complex ((MHC=major histocompatibility complex;HLA=human leukocyte antigen)). HLA genes are in a range of approximately4 million base pairs (bp) on human chromosome 6. So far attempts to makean unambiguous olfactory assignment of molecules to the development of aspecific individual odor have not been successful. However, there aresigns that MHC molecules play a causal role in the creation of aspecific individual odor profile (Proc. Natl. Acad. Sci. USA 94,2210–2214, 1997; Proc. Natl. Acad. Sci. USA 96, 1522–1525, 1999).

SUMMARY OF THE INVENTION

The object of this invention is to develop a perfume composition whichmakes it possible to produce odor-specific substances on the basis of aselection of certain alleles of olfactorily relevant genes andsubsequent treatment of their synthesis products; in the form of twoseparate formulations, these substances should then impart a differentbut mutual attractiveness to different partners.

According to this invention, a perfume composition is made availablewhich is produced by a method in which

-   a) of known HLA alleles of class I, an allele which differs by at    least one feature from other alleles of the HLA class I molecules is    selected, and which occurs in less. than 5% of the world's    population; or-   b) HLA alleles of class I produce by mutation of HLA alleles of    class I already present and which do not occur naturally and thus    have an incidence of 0% are selected; and-   c) the protein for which the selected allele codes is subjected to    an assembling operation in the presence of β₂-microglobulin (β₂m),    including single-chain constructs of HLA heavy chains of class I and    β₂m by forming a plurality of different peptides with a length of    typically 7 to 12 amino acids and with free N- and C-termini to form    soluble HLA class I molecules consisting of the extracellular    domains α₁, α₂ and α₃, β₂m and a peptide, whereby the peptide is    present in bound form in a peptide binding pocket of the HLA    molecule formed by the extracellular domains α₁, and α₂; and-   d) the HLA class I molecule thus formed with the bound peptide is    separated from the other constituents in a purification step; and-   e) the purified HLA class I molecule is subjected to fragmentation    with one or more proteases; and-   f) the substances that are active in forming the odor and are formed    in fragmentation are added as individual components or as a mixture    to the cosmetic preparation, optionally after first separating the    other substances.

To facilitate an understanding of this invention, the explanation ofintracorporeal formation of HLA class I molecules is given below.

It is known that mammals have the ability to differentiate between theirown tissue and that of another species as well as that of otherindividuals of their species. With the help of transplantation studiesin the mouse, different genes which are responsible for rapid rejectionof foreign tissue have been identified in the H-2 region on chromosome17. This gene complex has since then become known as the majorhistocompatibility complex (MHC). Human MHC is located on the short armof chromosome 6 and is known as HLA (human leukocyte antigen) complex.It contains, among other things, the genes for the MHC class I proteinsHLA-A, HLA-B and HLA-C, as well as HLA class II proteins. HLA moleculesbind intracellular peptides which are formed in degradation of cytosolic(typically MHC class I) or extracellular proteins (typically MHC classII) and transport them to the cell surface where HLA/peptide complexescan be recognized by the T-lymphocytes of the immune system. Other geneproducts coded in MHC are involved in the formation of peptide fragmentsand their transport.

HLA class I molecules consists of a membrane-anchoring heavy chain (43kDa) and a noncovalently associated light chain, β₂-microglobulin (β₂m;12 kDa; see FIG. 1). The heavy chain is composed of three extracellulardomains (α₁, α₂, α₃) each with approximately 90 amino acids, atransmembranal region approximately 25 amino acids long and a C-terminalcytoplasmic region. The asparagine preserved in position 86 of the α₁domains has undergone N-glycosylation in all HLA class I proteins.β₂-Microglobulin is not bound to membrane and has only one domain.

The three-dimensional structure of HLA class I molecules shown in FIG. 1is known from x-ray crystallographic studies (see, for example, J. Mol.Biol., Vol. 285, 645–653, 1999). β₂-Microglobulin and themembrane-proximal domains of the heavy chain (α₃) having thecharacteristic folding of molecules of the immunoglobulin superfamilysupport and stabilize the peptide bonding array formed by the α₁ and α₂domains in common, where the peptide is enclosed between two αZhelices,which are situated on a surface formed by one of eight antiparallelβZstrands (FIG. 3).

The heavy HLA chain is subject to a high polymorphism, but β₂m isidentical in all HLA class I molecules and is also coded on chromosome15 outside the HLA complex. So far approximately 70 HLA-A alleles,approximately 200 HLA-B alleles and approximately 70 HLA-C alleles areknown, each individual allele of which can express a maximum of twoalleles per gene locus. The variability of the alleles is limited almostcompletely to the α₁ and α₂ domains and concerns mainly amino acidswhose side chains point into the peptide bonding array or to the T-cellreceptor.

The bottom of the peptide bonding array consists of six differentlymanifested indentations or pockets which are formed by the amino acidside chains of the HLA protein. The peptides presented here usually havea length of eight or nine amino acids and are bound in an elongatedconformation (see FIG. 3). A preserved network of hydrogen ridgespositions the free N- and C-termini in the pockets at the ends of thebonding array and thus determines the orientation of the peptides.Additional allele-specific contacts exist between the side chains of thepeptide and the polymorphic amino acids of the HLA protein. The pocketsformed by the latter are capable of completely accommodating individualamino acid residues of the peptide which point downward into the bondingarray. These side chains are also referred to as “anchor residues”because they anchor the peptide securely in the bonding array. Each HLAprotein has at least one deep pocket formed by polymorphic amino acids,the pocket being specific for the respective allele. Only peptideshaving suitable anchor residues can be bound.

Both chains of an HLA class I molecule are synthesized separately in thecell and are conveyed cotranslationally into the endoplasmic reticulum(ER) where assembly of the heavy chain (HC, heavy chain) withβ₂-microglobulin (β₂m) and a peptide, to form functional complexes takesplace. A number of different proteins are involved in this coordinatedfolding process (Immunol. Today 21 (2000), 83–88).

This intracellular process can be completed in vitro in a similar mannerby causing the three components of the trimolecular end product, i.e.,an HLA class I molecule, to interact with one another in a suitablemedium. In this connection, it is possible to bring the DNA moleculesthat code for these three components to a expression in one, two orthree constructs so that the three components may be present eitherindividually or as a dimeric or trimeric protein for further processing.Of the known alleles, a selection is made such that preference is givento the rarest possible allele (i.e., occurring in less than 5% of theworld's population) over an allele occurring more commonly in thepopulation. The alleles HLA-A*6601 and HLA-B*7301 may be used as anexample because they occur extremely rarely (in less than 1% of thepopulation of Central Europe). It should be noted here that sequencedifferences between HLA class I alleles usually involve more than just asingle nucleotide position, so that the corresponding protein productsmay differ in a variable number of amino acids. These differencescharacteristically lead to differences in peptide binding behavior. Thetwo HLA molecules HLA-B35 and HLA-B53 may be used as an example becausethey differ only in the presence of the Bw6-(B35) and Bw4-(B53)determinants, i.e., in five altered amino acids. As a rule, the boundpeptides contain a tyrosine (B35) as the C-terminal anchor, whereseveral amino acids are tolerated as the C-terminus of the peptide ofHLA-B53 molecules. The other anchor in peptide position 2, however, is aproline in both molecules due to the identity of the molecules in theregion of the B pocket which binds this proline.

The relevant information for selecting alleles and peptides isaccessible to those skilled in the art and also to the general public(e.g., on the Internet at http://www.ebi.ac.uk/imgt/hla/ for DNAsequences and protein sequences of HLA molecules;ftp://ftp.wehi.deu.au/pub/biology/mhcpep/ orhttp//134.2.96.221/scripts/hlaserver.dll/home.htm for peptidesequences). Those skilled in the art can also deduce from this whichalleles have which features and at what frequency they occur.

Another embodiment of this invention consists of constructing artificialHLA class I alleles and synthesizing the respective proteins which donot occur in the human population according to the information currentlyavailable and which optionally have an altered peptide binding behavior.For example, it is possible through in vitro mutagenesis to replace theB pocket of the HLA-B27 molecule, which preferentially binds an argininein peptide position 2, with the B pocket of the HLA-A2 molecule, whichleads to binding of peptides having a hydrophobic amino acid such asleucine in the peptide position 2. The term “allele” therefore includesboth natural and artificial alleles.

After the selection, the soluble extracellular portion of theprotein(s), i.e., the α₁, α₂ and α₃ domains, are synthesized accordingto this invention and then in vitro assembly takes place in such amanner that in the presence of β₂m in a reconstitution solution at atemperature in the range of 0 to 40° C., the protein of the selected HLAallele is introduced into a peptide pool with different peptides of 7 to12 amino acids, preferably 8 to 9 amino acids, and incubated for aperiod of 1 to 7 days. The peptide pool contains in particular peptideshaving suitable “anchors” (anchor amino acids, see above) such asproline in position 2 and tyrosine in position 8 or 9 of a peptide inthe case of the HLA-B35 molecule. The octamer VPLRPMTY or the nonamerLPPLDITPY (J. Mol. Biol. 285, 645–653, 1999) may be used as such apeptide, for example.

The reconstituted trimeric HLA class I molecule is then separated bychromatography, e.g., by high-performance liquid chromatography (HPLC)or by FPLC from the other constituents of the mixture such as unboundpeptides, β₂m, etc. Correct folding of the reconstituted molecule andthus the native conformation are proven, e.g., with the help ofconformation-dependent antibodies in the ELISA test and by means ofimmunoprecipitation (Eur. J. Immunol., Vol. 23, 734–738, 1993; Hum.Immunol. 61, 408–418, 2000).

In a special embodiment of this invention, the HLA class I moleculeformed according to point c) or d) is placed in a mammalian serum,preferably a mouse serum, in particular a serum from β₂m(−/−) mice for aperiod of 1 to 36 hours, preferably 18 to 36 hours at 4° C. to 40° C.,preferably 37° C., before the subsequent fragmentation. Therefore,additional substances are optionally bound to the HLA molecule.

In subsequent fragmentation of the purified HLA class I molecule, aplurality of proteases may be used.

The proteases are preferably selected from proteases such as serineproteases, cysteine proteases, aspartate proteases and metal proteasesas well as peptidases such as amino peptidases, dipeptidases,dipeptidylcarboxypeptidases, carboxypeptidases, omega-peptidases. Forexample, pronase from Streptomyces griseus (Sigma Pronase type XIV) ispreferred (Proc. Natl. Acad. Sci. USA, Vol. 96, 1522–1525, 1999).

Fragmentation with pronase may take place, for example, at roomtemperature for two hours, whereby the concentration may vary as afunction of the experimental conditions.

For reconstitution of soluble HLA-A or HLA-B peptide complexes, areconstitution buffer may optionally be added, e.g., 400 mM L-Arg-HCl, 2mM EDTA, 5 mM reduced glutathione, 0.55 mM oxidized glutathione, 100 mMTris-HCl, pH 7.5.

After separation of the substances that are active in determining theodor, e.g., by chromatographic methods (HPLC, FPLC) or immunologicalmethods, these substances may be added to cosmetic preparations such asperfumes, emulsions, soaps, oils, gels, creams and the like inappropriate concentrations. Such formulations also form an object ofthis invention.

One particular feature of the perfume composition according to thisinvention is that it may consist of two separately formulated products.It is also possible to formulate three separate products.

In the preferred perfume combination according to this invention, theproduct produced according forms a combination unit with a secondproduct which is also produced by the method, with the provision thatthe selected allele for the second product is a different allele thanthe allele for the first product. The allele for the second product alsodiffers by at least one feature from other alleles of the HLA class Imolecules. This feature is a so-called rare feature which occurs in less5%, preferably less than 1% of the world's population (allelefrequency).

The combined unit includes two perfume formulations, one of which isprovided for or is to be assigned to different partners, preferablydifferent partners of different sexes.

Preferred perfume compositions are characterized in that the allelesHLA-A*6601 and HLA-B*7301; the alleles HLA-B*1301 and HLA-B*2709; theallele HLA-B*2705 with the replacement of the B pocket by the pocket ofthe HLA-A*0201 allele and HLA-B*7301 are used as starting materials forthe separate perfume compositions. These allele pairs form startingmaterials for corresponding perfume composition pairs, e.g., A and B,perfume A being assigned to partner A and perfume B being assigned topartner B to increase the attractiveness of partners A and B for oneanother.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention is illustrated in greater detail below by examples. Inthe respective drawings,

FIG. 1 shows a schematic diagram of an HLA class I molecule withextracellular domains α₁, α₂, α₃; the membrane-anchored heavy chain isnot covalently associated with β₂m.

FIG. 2 shows a structure of the extracellular fraction of an HLA class Imolecule.

FIG. 3 shows a view from above onto the peptide binding pocket havingthe peptide bound in a zigzag pattern (HLA-B*3501 with peptideLPPLDITPY).

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Producing Perfume Component A

Induction

LB medium=10 g bactotryptone, 5 g yeast extract, 10 g NaCl dissolved in1 L ddH₂O.

300 ml LB/amp medium was inoculated with 3 ml overnight culture (at roomtemperature) in a 1 L flask and agitated at 300 rpm at 37° C. untilreaching an OD₅₆₀ of 0.7. After adjusting the IPTG(IPTG=isopropyl-β-D-thiogalactopyranoside) concentration to 0.4 mM, thebacteria continued to grow for 4 more hours at 37° C. before beingcentrifuged for 20 minutes at 2700 g. The pellet was used forpreparation of inclusion bodies.

For identification of protein-expressing clones after transformation,induction was performed on a smaller scale. Portions of 200 μl LB/Ampwere inoculated with a colony in a 96-hole cell culture plate (Costar)and incubated overnight at room temperature. Then 100 μl was removed andplaced in a new well together with 100 μl LB/Amp/2×IPTG and left tostand for four hours at 37° C. Then the culture was centrifuged in anEppendorf vessel, the bacteria placed in a 100 μl SDS specimen buffer(SDS=sodium lauryl sulfate), lysed for 10 minutes at 95° C. and onealiquot was applied to an SDS polyacrylamide gel. Protein-expressingclones had an additional band.

Preparation of Inclusion Bodies

Foreign proteins are often deposited as a denatured precipitant inbacterial cytoplasm. These inclusion bodies can be purified by severalcentrifugation steps after cell digestion, dissolved in urea and thenreconstituted to yield functional proteins.

The pellet obtained after bacterial induction was dissolved in 10 mllysis buffer (25% sucrose, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0) and 1 mMphenylmethanesulfonic acid fluoride and frozen overnight at −20° C. orprocessed further immediately. After adding 0.5 ml lysozyme (10 mg/mllysis buffer) and incubating for 30 minutes on ice, the solution assumeda viscous consistency because of the release of DNA. Adding MgCl₂ (to 10mM), MnCl₂ (to 1 mM) and DNase I (to 10 μg/ml) caused the lysate to bereliquified. After centrifuging for 10 minutes at 10,000 g, the pelletwas resuspended by ultrasound in 10 ml detergent buffer (0.2M NaCl, 1%deoxycholic acid (Sigma), 1% Nonidet P-40 (Sigma), 2 mM EDTA, 2 mM DTT(dithiothreitol), 20 mM Tris-HCl, pH 7.5) and centrifuged again on iceafter incubating for ten minutes. The inclusion bodies were washed fourtimes in Triton buffer (100 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.5% TritonX100 (Serva), 50 mM Tris-HCl, pH 8.0) and dissolved in 4 ml 20 mMTris-HCl, pH 7.5, 150 mM NaCl, 10 mM DTT.

Reconstitution of the Functional HLA-peptide Complex

The functional molecule HLA A*6601 was reconstituted from the heavychain α₁, α₂, α₃ domains), dissolved in urea, β₂-microglobulin and thepeptide X

$\begin{matrix}T \\V\end{matrix}$XXXXXX

$\begin{matrix}R \\K\end{matrix}$according to the dilution protocol of Garboczi et al. (Proc. Natl. Acad.Sci. USA, Vol. 89, 3429–3433, 1992).

The subunits purified in the form of inclusion bodies were dissolved infreshly prepared and filtered urea buffer (50% urea, 50 mM NaCl, 20 mMTris-HCl, pH 7.5) (30 minutes at room temperature in an agitator), thencentrifuged (20 minutes, 10,000 g) and their concentration wasdetermined. The two chains were combined, topped off with urea buffer to5 ml and diluted in 200 ml reconstitution buffer (400 mM 1-arginine, HCl(Sigma), 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidizedglutathione, 100 mM Tris-HCl, pH 7.5) containing the peptide. The finalconcentration of the HLA chain was 1 μM and that of the β₂-microglobulinwas 2 μM and that of the peptide was 10 μM. The batch remained at 4° C.for 36 hours and was separated on a gel filtration column afterconcentrating to 1 ml with the help of Centriprep-10 concentration tubes(Amicon).

Gel Filtration

The HLA-peptide complex was purified by means of a Superdex 75-HR column(Pharmacia) equilibrated with 20 mM Tris-HCl, pH 7.5, 150 mM NaCl onwhich the concentrated restitution batch was separated at a flow rate of1 ml/min.

Fragmentation

1 mg of the purified HLA-peptide complex in 1 ml buffer (20 mM Tris-HCl,pH 7.5, 150 mM NaCl) is either mixed directly with bacterial pronasetype XIV from Streptomyces griseus (final concentration 0.1 mg/ml) oradded to serum from β₂m(−/−) mice that could no longer produce β₂mprotein because of defective genes for β₂m (final concentration of theHLA-peptide complex 1 mg/ml). Before adding the enzyme, induction of theHLA-peptide complexes may be performed in the serum at 4° C. to 40° C.for up to 24 hours; then it is mixed with bacterial pronase type XIVfrom Streptomyces griseus (final concentration 2 mg/ml). Degradation ofthe proteins is performed at room temperature for 2 hours. Otherconcentrations of the HLA-peptide complex and the enzyme as well as thedegradation time and temperature are possible. The fragmented proteinsare either frozen at −80° C. or processed further directly.

Separation

The components that are inactive with respect to scent may be separatedby means of Sephadex G-100 in a batch process where the specimencontaining the degraded proteins is mixed with Sephadex ( 1/10 of thevolume), incubated for 10 minutes at room temperature briefly. Thesupernatant contains the components of the specimen that have an activeodor effect.

This yields perfume component A.

EXAMPLE 2

Producing Perfume Component B

The procedure followed was the same as that described in Example 1 butthe allele used was HLA-B*7301 and as the peptide XRXXXXXXP. Thefollowing sets corresponded to those in Example 1, yielding perfumecomponent B after fragmentation and separation

EXAMPLE 3

Perfume Composition (eau de Perfume)

Composition A

At room temperature the following ingredients were mixed together (% byweight);

Perfume component A 11% Ethanol q.s. to 100 Water  1% Color blue 0.05%  

At the same time the following ingredients were mixed together:

Composition B

Perfume component B 10% Ethanol q.s. to 100 Water  1% Color yellow0.06%  

The two compositions were stored for 10 days at 5° C. to 10° C.thereafter and then packaged as a retail unit. Composition A was nextmade available to a partner of a committed relationship, and compositionB was made available to the other partner of the relationship. Next, thetwo partners each evaluated the perfume of the other partner as veryerotic and attractive.

EXAMPLE 4

Face and Body Cream

Part A

Phase A (in % by weight) Propylene glycol dicaprylate 4 Cetearylisononanoate 2.5 Shea butter 1.0 Dimethicone 1.2 Phase B Water q.s. to100 Glycerol 3 Phase C Preservative 0.5 Perfume component A 1.5 Orangedye 0.08

Phase A was heated to 70° C. and phase B was also heated separately to70° C. Then the two phases were mixed together and the mixture wascooled to 40° C. Next phase C was added and the entire mixture washomogenized.

Part B

The phase composition of phases A and B as in part A of Example 4 wasused.

Phase C Preservative 0.5 Perfume component B 1.5 Blue dye 0.08

These ingredients were processed like those for part A of Example 4.Parts A and B of the cream of Example 4 were finished together as oneretail unit. Then part A was made available to a partner of a committedrelationship and part B was made available to the other partner of therelationship. Each partner evaluated the perfume of the partner as veryattractive.

1. A perfume composition of HLA molecules prepared according to a methodwhereby a) an allele is selected from known HLA alleles of class I suchthat it differs by at least one feature from other alleles of the HLAclass I molecules which occurs in less than 5% of the world'spopulation; or b) HLA alleles of class I are selected which have beenproduced by mutation of existing HLA alleles of class I and do not occurnaturally and thus have an incidence of 0%; and c) the protein for whichthe selected allele codes is subjected to assembly in the presence ofβ₂-microglobulin (β₂m) by using a plurality of different peptides havinga length of typically 7 to 12 amino acids and with N- and C-termini toform soluble HLA class I molecules consisting of the extracellulardomains α₁, α₂ and α₃, β₂m and a peptide is formed, whereby the peptideis present in bound form in a peptide binding chain of the HLA moleculeformed by the extracellular domains α₁ and α₂; and d) the HLA class Imolecules thus formed together with the bound peptides are separatedfrom the other constituents in one purification step; and e) thepurified HLA class I molecules are subjected to fragmentation with oneor more proteases; and f) the substances that are active in producingthe odor and are formed in fragmentation are added either as anindividual component or as a mixture to a cosmetic preparation,optionally after first separating the other substances.
 2. The perfumecomposition according to claim 1, wherein the proteases are selectedfrom proteases such as serine proteases, cysteine proteases, aspartateproteases and metal proteases, amino peptidases, dipeptidases,dipeptidyl carboxypeptidases, carboxypeptidases, omega-peptidases. 3.The perfume composition according to claim 1, wherein the protease ispronase.
 4. The perfume composition according to claim 1, wherein thealleles HLA-A*6601 and HLA-B*7301, the alleles HLA-B*1301 andHLA-B*2709, the alleles HLA-B*2705 with the replacement of the “B”pocket by the “B” pocket of the HLA-A *0201 allele and HLA-B*7301, areused as starting materials for separate perfume composition pairs. 5.The perfume composition according to claim 1, wherein the HLA class Imolecule formed according to point c) or d) is added to a serum from β₂m(−/−) mice for a period of 1 to 36 hours at 4° C. to 40° C. beforesubsequent fragmentation.
 6. The perfume composition according to claim1, wherein t the product produced forms a combination unit with a secondproduct which is produced with the provision that the selected allelefor the second product is a different allele from the allele for thefirst product, the allele for the second product also differs by atleast one feature by other alleles of the HLA class I molecules, and itoccurs in less than 5% of the world's population.
 7. The perfumecomposition according to claim 6, wherein it is in the form of at leastone of a clear solution, soap, cosmetic gel, cosmetic emulsion, and acream, in a concentration of 0.0001 to 20 wt %, based on the totalcomposition.
 8. A method for, said method comprising administeringtopically to each of the partners a different perfume composition of HLAmolecules selected from a perfume composition pair, each perfumecomposition prepared according to a method whereby a) an allele isselected from known HLA alleles of class I such that it differs by atleast one feature from other alleles of the HLA class I molecules whichoccurs in less than 5% of the world's population; or b) HLA alleles ofclass I are selected which have been produced by mutation of existingHLA alleles of class I and do not occur naturally and thus have anincidence of 0 %; and c) the protein for which the selected allele codesis subjected to assembly in the presence of β₂-microglobulin (β₂m) byusing a plurality of different peptides having a length of typically 7to 12 amino acids and with N- and C-termini to form soluble HLA class Imolecules consisting of the extracellular domains α₁, α₂ and α₃, β₂m anda peptide is formed, whereby the peptide is present in bound form in apeptide binding chain of the HLA molecule formed by the extracellulardomains α₁ and α₂; and d) the HLA class I molecules thus formed togetherwith the bound peptides are separated from the other constituents in onepurification step; and e) the purified HLA class I molecules aresubjected to fragmentation with one or more proteases; and f) thesubstances that are active in producing the odor and are formed infragmentation are added either as an individual component or as amixture to a perfume preparation, optionally after first separating theother substances.
 9. The method as in claim 7, wherein the first partneris assigned the allele HLA-A*6601 and the second partner HLA-B*7301; thefirst partner is assigned the allele HLA-B *1301 and the second partnerHLA-B*2709; or the he first partner is assigned allele HLA-B*2705 withthe replacement of the “B” pocket by the “B” pocket of the HLA-A*0201allele and the second partner HLA-B*7301.